There has been a growing interest in the application of microfluidic systems to a variety of technical areas, including such diverse fields as biochemical analysis, medical diagnostics, analytical chemistry, chemical synthesis, and environmental monitoring. Microfluidic systems provide certain advantages in acquiring chemical and biological information. For example, microfluidic systems permit complicated processes to be carried out using very small volumes of fluid, thus minimizing consumption of both samples and reagents. Chemical and biological reactions occur more rapidly when conducted in microfluidic volumes. Furthermore, microfluidic systems permit large numbers of complicated biochemical reactions and/or processes to be carried out, either in parallel or in series, within a small area (such as within a single integrated device) and also facilitate the use of common control and interface components.
Among the various branches of analytical chemistry, the field of chromatography stands to particularly benefit from the application of microfluidic technology due to higher efficiency and increased throughput, such as may be afforded by performing multiple analyses in parallel in a miniaturized format. Chromatography encompasses a number of methods that are used for separating closely related components of mixtures. In fact, chromatography has many applications including separation, identification, purification, and quantification of compounds within various mixtures.
Liquid chromatography is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a “stationary phase” material (e.g., packed particles having functional groups and disposed within a tube) commonly referred to as a “separation column.”) A sample is supplied to a separation column (stationary phase material) and carried by the mobile phase. As the sample solution flows within the mobile phase through the stationary phase, components of the sample solution will migrate according to interactions with the stationary phase and these components are retarded to varying degrees. The time a particular component spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following chromatographic separation in the column, the resulting eluate stream (consisting of mobile phase and sample components) contains a series of regions having elevated concentrations of individual species, which can be detected by various techniques to identify and/or quantify the species.
Although pressure-driven flow or electrokinetic (voltage-driven) flow can be used in liquid chromatography, pressure-driven flow is desirable since it permits a wider range of samples and solvents to be used and it avoids problems associated with high voltage systems (such as hydrolysis, which can lead to detrimental bubble formation). Within pressure-driven systems, higher pressures generally provide greater separation efficiencies. In conventional high performance liquid chromatography (“HPLC”) systems, pressures of several hundred to thousands of pounds per square inch (psi) are commonly used with densely packed separation columns to provide increased separation efficiency and reduced separation times. A standard liquid chromatography column for performing HPLC has a tubular column body (typically steel, although materials like glass, fused silica, and/or PEEK are also used) and a high precision internal bore containing packed (particulate) stationary phase material, with the tube bounded at either end with fine filters or “frits” and end fittings. Samples are separated serially (i.e., one at a time) in conventional columns, which are re-used several times (and flushed between each use) before they become so contaminated that their effectiveness is diminished.
Typically, eluate from a separation column will be subjected to one or more analytical processes, including, but not limited to, optical detection, such as ultraviolet/visible (“UV/Vis”) light absorbance or refractive index detection. To facilitate optical detection, a flow cell having optical windows and an eluate path therethrough is disposed downstream of the chromatography column, with fluid connections made by way of threaded fittings. A common problem that can interfere with post-column optical detection in HPLC systems, however, is the formation of bubbles in the detection region. Such bubbles are formed as gases dissolved in the mobile phase expand downstream of the column in the lower pressure environment of a flow cell, often causing baseline noise and drift. To mitigate such problems, conventional HPLC systems utilize solvent degassers upstream of a separation column to reduce the presence of gas in mobile phase, and further employ means to increase the backpressure in a detection region (such as a flow cell) downstream of the separation column.
Two general approaches are used to increase backpressure in a detection region: either providing a backpressure regulator (valve) or providing a capillary restrictor (tube) of appropriate length downstream of the detection region. Backpressure regulators are advantageously capable of providing a constant backpressure in a detection region over a range of different operating pressures. The utility of such regulators is limited, however, because they are expensive and require multiple parts that may reduce their reliability. Such regulators are typically characterized by non-negligible internal dead volume, which may “smear” or otherwise distort bands of species flowing therethrough, thus limiting the ability to perform further desirable analytical techniques (such as, for example, mass spectrometry) downstream of the optical detection region. Additionally, due to their expense and size, it would be difficult and/or impractical to use conventional HPLC backpressure regulators with highly parallel microfluidic separation systems.
The second conventional approach to elevating backpressure, providing a capillary tube downstream of an optical detection region, is inexpensive and involves no moving parts. But the utility of capillary restrictors is also limited due to packaging constraints and other concerns. To provide a desired backpressure, a capillary of appropriate size should be selected for a specific column exit condition (i.e., pressure and flow rate). Backpressure is generally increased by reducing the inner diameter of a capillary (but exceedingly small capillaries can become clogged) and/or increasing the length of the capillary (but very long capillaries can interfere with packaging constraints). In highly parallel microfluidic separation systems, it may be very cumbersome to provide one capillary restrictor for each separate detection region due to packaging constraints and the difficulty of making a large number of reliable, fluid-tight connections to small-bore capillary tubes.
In light of the foregoing, it would be desirable to provide a separation device having an optical detection region with compact means for elevating backpressure within the detection region. It would be further desirable for such a system to be inexpensive and easy to fabricate, with a minimum of moving parts to promote reliable, leak-free operation.